There is a need for a reliable in vitro tumor model system for testing the effect of chemical or physical (such as radiation or heat) treatment on tumor cells and the effect of such treatment on surrounding normal cells, which would accurately reflect the effect of such treatment on an actual in vivo tumor. At present, soft agar stem cell techniques are known as in vitro tumor assay methods. See A.W. Hamburger et al., "Direct Cloning of Human Ovarian Carcinoma Cells in Agar," 38 CAncer Res. 3438-3447 (1978). However, soft agar assay methods have several limitations and drawbacks: (a) not all tumor biopsy samples yield colonies which are suitable for soft agar assays; (b) the plating efficiency of this system is very low (ca 10.sup.-5) and appears not to be typical of in vivo tumor clonogens; and (c) the system is not suitable for radiation studies. In addition, the systems predictive ability is also limited.
Attempts have also been made to use multicellular tumor spheroids (MTS) systems for assaying tumor inactivating agents. See, J.M. Yuhas et al., "In Vitro Analysis of the Response of Multicellular Tumor Spheroids Exposed to Chemotherapeutic Agents in Vitro or in Vivo," 38 Cancer Res. 3595-3598 (1978).
MTS systems are desirable as in vitro models because some of their features more closely resemble those of actual in vivo tumors than those of the soft agar techniques. See R.M. Sutherland, "Cell and Environment Interaction in Tumor Microregions: The Multicell Spheroid Model." 240 Science, 177-184 (1988); R.E. Durand, "Cure, Regression and Cell Survival: A Comparison of Common Radiobiological Endpoints Using an in Vitro Tumor Model," 48 British Journal of Radiology, 556-571 (1975). For example, often in both spheroids and in tumors there is an increased survival rate for the cells when they are maintained in close contact during and after radiation treatment. The differences in survival rates of cells in spheroids are attributed to several factors, including hypoxia in spheroids as well as the three-dimensionality of cellular contact within these bodies. See, R.P. Hill et al., "The Effect of Intercellular Contact on the Radiation Sensitivity of KHT Sarcoma Cells," 77 Radiation Res 182-192 (1979); G.M. Hahn et al., "Repair of Potentially Lethal Damage in Vivo in Solid Tumor Cells After X-radiation,"34 Cancer Res. 351-354 (1974); R.E. Durand and R M. Sutherland, "Effects of Intercellular Contact on Repair of Radiation Damage," 71 Exp. Cell Res. 75-80 (1972); H. Gershman, J. Drumm and L. Culp, "Sorting of Normal and Virus-Transformed Cells in Cellular Aggregates," 68 Journal of Cell Biology, 276-286 (1976); P.J. Tofilon, N. Buckley and D.F. Deen, "Effect of Cell-Cell Interactions on Drug Sensitivity and Growth of Drug-Sensitive and Resistant Tumor Cells in Spheroids," 226 Science 862-864 (1981).
The use of presently available MTS assay systems however, cannot be applied to all test cells, such as tumor cells, because not all test cells are capable of forming spheroids. For example, A.C. Jones et al., "In Vitro Cytotoxic Drug Sensitivity Testing of Human Tumor Xenografts Grown as Multicellular Tumor Spheroids," 46 Br. J. Cancer 870-879 (1982), report that none of the cells taken directly from tumors of seven patients could form spheroids, while only 5 out of 22 cell lines grown as xenografts could do so. Thus, at present, use of MTS systems for the study of tumor inactivating agents is restricted to those cells which are capable of forming spheroids. In addition, because the present MTS assay system is tailored to the particular test cell type which comprises the MTS system, MTS studies are not conducted under uniform conditions and, thus, do not yield uniform, comparable results.